This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Purification Normal Phase Chromatography Sample was dissolved in butanol:ethanol:water 4:1:1 and loaded onto a 2ml column of Avicel microcrystalline cellulose then eluted with 7ml of the above solvent. Sample was eluted with 6ml ethanol:water 1:1. Most of the color and carbohydrate eluted in this stage as judged by phenol sulfuric acid assay while most of the salt eluted in the load. Reverse Phase Chromatography Material from the 50% ethanol wash of the normal phase column was further purified by loading onto a Waters C18 Sep Pak in 5% methanol. 1ml of 5% methanol was then run through the column and 4ml 50% methanol was used for elution. All color and most carbohydrate eluted in this stage. Glycosyl linkage NaOH method: For glycosyl linkage analysis, the sample was permethylated, depolymerized, reduced, and acetylated;and the resultant partially methylated alditol acetates (PMAAs) analyzed by gas chromatography-mass spectrometry (GC-MS) as described by York et al (1985) Methods Enzymol. 118:3-40. Initially, an aliquot of sample was permethylated by the method of Ciucanu and Kerek (1984) Carbohydr. Res. 131:209-217 (treatment with sodium hydroxide and methyl iodide in dry DMSO). This involved incubating the sample in 1M NaOH in DMSO for 10 minutes at room temperature then adding 400ul of methyl iodide and incubating 10 more minutes. 80ul of 2.0M NaOH in DMSO were then added and after 10 more minutes 80ul of methyl iodide were added. 40 minutes after this, 80 ul of NaOH were added and allowed to incubate 40 minutes whereupon 2ml of water were added and the methyl iodide evaporated by bubbling. Following sample workup, the permethylated material was hydrolyzed using 2 M trifluoroacetic acid (2 h in sealed tube at 121 degrees C), reduced with NaBD4, and acetylated using acetic anhydride/trifluoroacetic acid. The resulting PMAAs were analyzed on a Hewlett Packard 5890 GC interfaced to a 5970 MSD (mass selective detector, electron impact ionization mode);separation was performed on a 30 m Supelco 2330 bonded phase fused silica capillary columnInitially, an aliquot of sample was permethylated by the method of Ciucanu and Kerek (1984) Carbohydr. Res. 131:209-217 (treatment with sodium hydroxide and methyl iodide in dry DMSO). The permethylation was repeated twice in order to aid complete methylation of the polymer. Following sample workup, the permethylated material was hydrolyzed using 2 M trifluoroacetic acid (2 h in sealed tube at 121 degree C), reduced with NaBD4, and acetylated using acetic anhydride/trifluoroacetic acid. The resulting PMAAs were analyzed on a Hewlett Packard 5890 GC interfaced to a 5970 MSD (mass selective detector, electron impact ionization mode);separation was performed on a 30 m Supelco 2330 bonded phase fused silica capillary column. NMR Spectroscopy The sample was deuterium-exchanged by lyophilization from D2O (99.9 % D, Aldrich) and dissolved in 40 uL D2O (99.996 % D, Cambridge Isotope Laboratories). 1-D Proton and 2-D gradient HSQC, and TOCSY NMR spectra were acquired in a capillary probe on a Varian Inova-600 MHz spectrometer at 298 K (25 [unreadable]C). Proton chemical shifts were measured relative to the HDO signal (delta=4.60 ppm at 40 [unreadable]C).